The long-term goal of our research is to dissect the mechanism of iron uptake and the regulation of its expression in the pathogens Vibrio anguillarum and V. vulnificus. In the fish pathogen V. anguillarum the pJM1 mediated iron uptake system plays an essential role in systemic infection. This system is an excellent model due to its striking similarities to human septicemic diseases, such as those caused by V. vulnificus in iron-overloaded patients. Even under these high iron conditions, there are iron-limiting niches at which iron limitation-induced genes play a vital role in virulence. In this competitive renewal we will complete the analysis of the regulation of the plasmid-mediated iron uptake genes of V. anguillarum and dissect the mechanism of iron transport in V. anguillarum and V. vulnificus. We have previously characterized the essential components of the V. anguillarum iron uptake system: the siderophore anguibactin and the proteins and regulatory elements controlling its synthesis and transport. We also characterized iron-regulated genes crucial to the virulence of V. vulnificus. We propose: 1. Mechanism of regulation of the ITB operon by AngR and RNAb. AngR is a bifunctional protein that intervenes in anguibactin biosynthesis and acts in the regulation of expression of the iron transport-biosynthesis (ITB) operon. We intend to: a) complete the analysis of the regulatory domains of AngR and b) characterize the structure-function of RNAb in the regulating transcription within the ITB operon. 2. Molecular characterization of the mechanisms of TonB2- mediated iron transport and virulence in V. anguillarum. Of the two tonB systems available to transduce the energy of the proton motive force of the cytoplasmic membrane of V. anguillarum, only the tonB2 system is required for virulence. Since we recently solved the structure of the TonB2 protein, we intend to identify residues important for the TonB2 recognition of the FatA outer membrane receptor and to identify how specific mutations in FatA affect virulence. 3. Characterization of the TtpC function in the TonB2-mediated iron transport and virulence in V. anguillarum and V. vulnificus. We identified a gene upstream of tonB2 in V. anguillarum, V. vulnificus, and V. cholerae that encodes a novel protein essential for tonB2-mediated iron transport. We will characterize this protein, TtpC, to understand its role in iron transport and virulence of V. anguillarum and V. vulnificus.